Synthetic fermentation medium and process using same for cultivating gibberella zeae

ABSTRACT

A SYNTHETIC FERMENTATION MEDIUM, PARTICULARLY FOR USE IN THE PRODUCTION OF AN ANABOLIC AND ESTROGENIC SUBSTANCE IDENTIFIED IN U.S. PAT. NO. 3,196,019, BY CULTIVATON OF THE MICROORGANISM GIBBERELLA ZEAE (GORDON). THE MEDIUM INCLUDES EXFOLIATED VERMICULITE AS A SUPPORT FOR THE MICROORGANISM AND AN AQUEOUS NUTRIENT PRODUCTION MEDIUM CONTAINING A SUGAR SUCH AS GLUCOSE AS THE CARBOHYDRATE SOURCE FOR THE MICROORGANISM.

United States Patent 3,580,811 SYNTHETIC FERMENTATION MEDIUM AND PROCESSUSING SAME FOR CULTIVATING GIBBERELLA ZEAE Phil Hidy, Terre Haute, Ind.,assignor to Commercial Solvents Corporation No Drawing.Continuation-impart of application Ser. No. 670,455, Sept. 25, 1967.This application Apr. 16, 1968, Ser. No. 721,606

Int. Cl. C1211 3/02; C126 13/00 US. Cl. 195--100 21 Claims ABSTRACT OFTHE DISCLOSURE A synthetic fermentation medium, particularly for use inthe production of an anabolic and estrogenic substance identified in US.Pat. No. 3,196,019, by cultivation of the microorganism Gibberella zeae(Gordon). The medium includes exfoliated vermiculite as a support forthe microorganism and an aqueous nutrient production medium containing asugar such as glucose as the carbohydrate source for the microorganism.

This application is a continuation-in-part of copending application Ser.No. 670,455, filed Sept. 25, 1967, now abandoned.

This invention relates to improved methods for the production andrecovery of an anabolic and estrogenic substance such as is described inUS. Letters Patent 3,196,019, i.e., a compound having the structuralformula:

The compound described above is, as described in U.S. Pat. No.3,196,019, produced by cultivating the microoragnism Giberella zeae(Gordon) on a suitable nutrient medium and will be hereinafter describedas F.E.S., standing for Fermentation Estrogenic Substance. In producingF.E.S. the spores or the vegetative mycelia of the microorganism areincubated in a suitable inoculation medium. Heretofore, themicroorganism-containing inoculation medium has then been introducedinto a fermentation medium containing one of the common grains as thecarbohydrate source, i.e., finely divided corn. After a fermentationperiod of about 6 to 20 days, or more, the F.E.S. produced can berecovered from the fermentation medium, e.g. as described in US. Patent3,196,019, by extracting the fermentation medium with 95% ethanol,concentrating the ethanol extract, dissolving the concentrated extractin chloroform, extracting the chloroform solution with sodium carbonatesolution having an adjusted pH of about 912, and then acidifying thesodium carbonate extract with hydrochloric acid to a pH of about 6 to6.5 to precipitate the solid impure F.E.S.

A method for production of PBS. utilizing synthetic fermentation mediais now provided in accordance with this invention whereby improvedconversions of carbohydrate to F.E.S. are realized and improved methodsof recovery of F.E.S. from the fermentation media can be used wherebyimproved overall yields of F.E.S. can be obtained.

In general the synthetic fermentation medium of this invention includesan inert support material and an aqueous production medium containing asan assimilable carbohydrate source a' sugar such as glucose forinstance.

3,580,811 Patented May 25, 1971 Other nutrients in the fermentationmedium advantageously include an assimilable nitrogen source as well asminerals, for instance sodium, potassium, magnesium, ammonium, chlorideand phosphate ions.

The support material is preferably a material which is inert to theorganism, insoluble in the recovery process solvents, and has a porositysufiicient to absorb the production medium. Exfoliated vermiculite whichhas such desirable properties as high porosity, high void volume tosurface area ratio, low density, relative chemical inertness andavailability in a wide range of particle sizes is the preferred supportmaterial. Vermiculite is a micaceous mineral generally identifiedchemically as a hydrated magnesium-aluminum-iron silicate, with aplatelet-type, or laminar, crystal structure which exfoliates whenheated or subjected to certain chemical reactions. The mineral occursnaturally in an unexpanded state and is mined in conventional manners.The largest domestic deposits of vermiculite are located in Montana andSouth Carolina. Chemical analyses of vermiculite from the two areas, seeTable I, indicate that they are essentially the same.

TABLE I Percent The mined ore is milled to controlled sizes and thenexfoliated. Exfoliation is generally accomplished by dropping the milledore in a continuous stream through a furnace, the chamber temperature ofwhich is variously reported as from about 1400 to 2200 F. Duringexfoliation, each granule or particle or vermiculite expands to severaltimes its original size and traps within itself thousands of air cells.The exfoliation of vermiculite crystals results in large pores beingformed between groups of platelets. Thus, exfoliation makes available alarge increase in void volume without significantly changing the surfacearea of the latelets.

In general, the smaller the particle size of the exfoliated vermiculiteused in this invention the greater the yields of F.E.S. The particlesshould not, however, be so fine as to make filtration ditficult whichwould complicate F.E..S. recovery. The smaller particles are preferredsince they will hold more liquid, i.e. aqueous production medium,without actually becoming wet whereas in larger vermiculite particlesthe space between the vermiculite particles becomes so large that itcannot physically support the mycelium which is the vegetable growth ofthe microorganism. Particles which pass a US. Sieve Series, sieve No. 1,and are retained by sieve No. are suitable for this invention. Insmaller fermentations, that is those using less of the synthetic medium,the smaller sizes are actually preferred since, although filtrationdifficulties may be increased. the total amount of vermiculite beingworked is less which tends to alleviate these difficulties.Additionally, in smaller fermentations, the support is generallyprovided in thinner layers so that heat transfer and oxygen transferthroughout the support is easier than in larger fermentations.

The bulk density of exfoliated vermiculite is a general measure of theporosity of exfoliated vermiculite which is one of its desirablefeatures. The lower the bulk density, the greater the porosity of thevermiculite for a given particle size. Although the bulk density canvary for a given particle size, it is generally known that the bulkdensity of exfoliated vermiculite diminishes as the temperature ofexfoliation is increased until a level is reached after which there ispractically no further reduction in density. It is also known that thefriability, that is the tendency of the exfoliated vermiculite tomechanically break down in handling, increases with increasingtemperature. In general, the bulk density of the exfoliated vermiculiteused in this invention should not be so small that the vermiculiteparticles collapse upon application of the liquid production medium, orupon being transported, and should not be so high that the porosity ofthe vermiculite is insufficient to support the desired amount of aqueousproduction medium. Exfoliated vermiculite hav ing bulk densities ofabove 4 pounds per cubic foot, or about 5 to 10, or even 12 pounds percubic foot are suitable for use in this invention, although preferablythe bulk density is less than about 8 or 9 pounds per cubic foot and isparticularly in the range of about 5 to 7 pounds per cubic foot.Exfoliated vermiculite is commercially available in several grades ofsieve size and bulk density including those marketed by W. R. Grace andCompany under the trade names Zonolite, Verxite or Terralite which arerespectively, insulation grade, pure chemical grade and agricultralgrade vermiculites. Several grades of these commercially availableexfoliated vermiculites are identified in Table II by their averagedensity and sieve analysis.

TABLE II.SIE"E ANALYSIS, STANDARD GRADES Cumulative percent U.S. std.retained I sieve Grade N0. size Maximum Minimum l 0 1 Density: 4 to 7lbs. per cu. ft. 4 60 30 Sieve analysis (by weight) 8 95 65 16 100 85 40 2 Density: 4 to 8 lbs. per cu. it. 8 80 0 Sieve analysis (by weight)16 90 5 30 100 0 8 10 0 3 Density: 5 to 9 lbs. per cu. it. 16 G0 20Sieve analysis (by weight) 3O 95 65 50 98 75 100 100 00 16 5 0 4Density: 6 to 10 lbs. nor on. it. 05 16 Sieve analysis (by weight) 98100 100 The amount of inert support material normally desired will varydepending up the particular container being used. Factors involved inselection of the amount of support material are the requirement of themicroorganism for air and the necessity to remove carbon dioxideproduced with the F.E.S. Additionally, the temperature of the mediumshould be controlled within narrow limits during F.E.S. production.Accordingly, normally a relatively thin layer, e.g. about 1 or 1 /2inches to 6 inches, preferably about 3 to 5 inches, or less, ofsynthetic me dium is preferred.

The assimilable carbohydrate source in the aqueous fermentation mediumis advantageously sugar, preferably a monoor di-saccharide such asglucose, sucrose or maltose and is present in the aqueous medium inamounts suflicient for reduction by the microorganim to produce PBS. andbelow the amount at which it begins to crystallize out of the medium.Generally, the carbohydrate, e.g. glucose, is present in amounts fromabout 20% to about 50% by weight of the production medium, preferablybetween about 30 and 45% by weight. In addition to the carbohydratesource, nutrient media utilized also require the presence of a nitrogensource in either organic or in organic form such as urea, ammonium saltssuch as ammonium sulfate, ammonium nitrate, ammonium chloride, ammoniumtartrate, etc. Also, a source of potassium and phosphorus such asdipotassium phosphate, for instance, is advantageously used for suitableyields as are trace minerals containing such minerals as manganese,magnesium, iron, etc., in compounds such as magnesium sulfate, ferroussulfate, manganese sulfate, etc. The amounts of the various minerals canvary considerably although each should be present in an amountsufficient to insure proper growth of the microorganism. Generally,amounts of the minerals suflicient to provide the desired ions in themedium in amounts of about 0.001 to 1% by weight are sufficient.Preferred minerals, or ions, and amounts are potassium (K+), about 0.01to 1.0% by weight, phosphorus (as HPO about 0.01 to 1% by Weight,particularly about 0.15 to 0.5%; and sodium nitrate and ammoniumnitrate, each in amounts of from about 0.1 to 3 or 5%. Yeast is alsoincluded in the aqueous production medium in an amount sufiicient toprovide the necessary growth factors, e.g. B vitamins, for themicroorganism. Preferably, distilled water, or deionized water, is usedin the production medium although tap water can be used if reducedyields of F.E.S. are acceptable. A basic production medium suitable foruse with this invention is illustrated in Table III with the percentageslisted being grams per cc. to make up the final medium.

To form the synthetic fermentation medium, the aqueous production mediumis advantageously added to the inert support material in an amount suchthat the support material does not become soggy but is moistsubstantially throughout each support particle. Generally, about 240milliliters of production medium is sufficient for from about 40 to 240grams of the support material, and preferably is used with about 80grams. In covered fermentation trays containing a vermiculite layerabout 3 inches deep and having surface dimensions of 20 by 40 inches,the production medium can be added in amounts up to about 19 or 20liters per tray. Since, if a major portion of the vermiculite remainsdry, the liquid will distribute so thinly that the microorganism willnot grow properly, it is generally preferred to use above about 8 to 10liters of production medium per tray. One factor affecting the desiredamount of liquid medium is the bulk density of the exfoliatedvermiculite. For example, in trays as described above, with the No. 2grade vermiculite identitied in Table II, optimum production of PBS. isobtained with about 15 liters of production medium per tray whereas witha No. 3 grade exfoliated vermiculite, optimum production is obtainedwith about 17 liters of production medium per tray.

F.E.S. is readily produced by cultivating the organism Gibberella zeae(Gordon) on the synthetic fermentation medium of this invention. A liveculture of the organism is on deposit with the United States Departmentof Agriculture, Agricultural Research Service, Northern UtilizationResearch and Development Division, Peoria, Ill. under the number NRRL3311. In carrying out the fermentation, an inoculum of the organism isprepared and mixed with the aqueous nutrient production medium fordistribution throughout the inert support to provide the fermenationmedium. If desired, the inoculum, medium and support can be mixed andthen spread over the fermentation tray. During fermentation, thetemperature of the fermentation medium is preferably controlled toprovide for optimum production of F.E.S. by placing the fermentationcontainer in a temperature controlled bath, such as a water bath,maintained generally at a temperature of from about 12 to 25 C.,preferably about 14 to 19 C., particularly about 15 to 17 centigrade.The pH of the fermentation medium should be between about 4 /2 and 6 /2,preferably between 5 and 6. Under these conditions, a suitable yield ofF.E.S. can be obtained in a period ranging from about 1 to 6 weeks,usually 4 to 5 weeks, depending upon the vitality of the microorganism.Recovery of the PBS. so produced can be accomplished by the methoddescribed in US. Pat. 3,196,019 although a preferred recovery process isdescribed in applicants copending application Ser. No. 670,393 filedSept. 25, 1967.

The following examples serve to further illustrate this invention:

EXAMPLE I Inoculum preparation Spores from a well sporulated Bennettsagar slant culture of the organism Gibberella zeae, strain NRRL 3311, nomore than two months old are suspended in 5 ml. of sterile water addedto the slant tube and are transferred to a SOO-ml. Erlenmeyer flaskcontaining 100 ml. of sterile Bennetts medium. The inoculated flask isshaken on a rotary shaker at 30 C. for 24 hours. Ten m1. of the firststage inoculum is used to inoculate 300 ml. of sterile Bennetts mediumin a l l. Erlenmeyer flask equipped with a side arm at the bottom of theflask to which is attached a length of rubber tubing having a shortglass tube inserted at the terminus. The tube is covered with a milkfilter disc and with paper to keep it sterile after sterilization. Allflasks are closed with loose cotton plugs. The inoculated second stageis shaken on a reciprocal shaker at 30 C. for 24 hours.

EXAMPLE II Fermentation procedure Fermentations are conducted inaluminum trays (5 x 20 x 40 inches) covered by aluminum lids (2 x 20.5 x40.5 inches) resting on six laboratory tubing clamps spaced equally andattached to the upper edge of the tray. This supports the under-surfaceof the lid about 0.25 inch above the upper edge of the tray.

The trays are filled to a level depth of 3 inches with exfoliatedvermiculite; the lids are put in place; and the trays are autoclaved forabout two hours at p.s.i.g. In the control trays an insulation gradeexfoliated vermiculite (broad, coarsely screened, mixed #1, #2, #3grades Zonolite described in Table II above) is used.

A production medium for the fermentation is prepared according to theformula below.

Cerelose is a commercially available glucose monohydrate. Allpercentages are grams per 100 cc. to make up the final medium. Thirteenand one-half liter quantities are dispensed into S-gallon stainlesssteel milk cans, the lids of which have been equipped with two stainlesssteel tubulatures, one of which just enters the lid and serves as an airinlet and the other of which extends to the bottom of the can with abend which places the bottom orifice near the outer edge of the can.Lids are seated firmly in place. One end of a short piece of rubbertubing is attached to the air tubulature, the other to a piece of glasstubing in which is lightly packed a wad of glass wool to serve as an airfilter. A longer piece of tubing is attached to the remainingtubulature. The glass air-filter and the end of the longer rubber tubingare covered with a milk filter disc and a paper outer covering and thecan and contents are autoclaved 20 min. at 15 p.s.i.g.

The sterile production medium is inoculated by introducing the contentsof one second-stage inoculum flask, prepared according to Example I,through the longer rubber tubing. Inoculum is thoroughly dispersed inthe medium and the contents of one can are dispensed evenly through thelonger rubber tube over the surface of the vermiculite in one tray.Siphoning of can contents is started by forcing air into the airtubulature.

The loaded trays are immersed to a depth of about two inches in a pancirculating water at an entrance temperature of 15l7 C. The circulationrate is adjusted so that the heat generated by the fermentation processdoes not increase the exit water temperature by more than one degreecentigrade.

Incubation is continued for from two to six weeks, or until theconcentration of F .E.S. reaches a maximum.

Sampling is done weekly by removing 15 spaced plugs 2 cm. in diametercut through the entire depth of the cake. The plugs are crumbled byrubbing through a screen, mixed thoroughly and a g. sample is assayed.

In the assay, one hundred grams of cake is extracted two times withwater by grinding for about 30 seconds in a Waring Blendor. Each extractis removed by vacuum filtration. Combined Water extracts total about600-700 ml. Water extracted samples are air dried under heat lamps andare extracted three times with methanol in the same fashion. Combinedmethanol extracts total about 700-800 ml.

Methanol extracts are diluted with spectroscopic grade methanol tocontain 5-10 ,ug. F.'E.S. per ml. and the absorbance of the dilutesolution is compared at 236 mp. with a standard solution of pure RES. inspectroscopic methanol prepared to contain 10 g. RES. per ml. Absorbanceat 236 m, is directly proportional to concentration.

Results from a typical control fermentation carried out as describedabove are as follows:

F.E.S. (g./tray) for 3 weeks 4 weeks 5 weeks EXAMPLE HI were used. TableIII illustrates variables in the production medium and their effect uponthe amount of RES. produced.

TABLE III Variable FES percent of control Four times the amount of yeast90, 82 Omit yeast, FeSO and KCl 76 Omit yeast and FeSO 83 Omit yeast andKCl 80 Omit FeSO and KCI 96 Replace K with NH,+ 17 Replace K+ with Na+28 lReplace Na+ with K+ 36 Replace S and Cl" 91 Two times the amount ofNaNO 73 Two times the amount of K2HPO4 73 Two times the amount of PC, asKH PO 73 Two times the amount of SO as MgSO -+K SO' 14 Two times theamount of MgSO 33 Add 21101 05%) 12 Replace KCl with ZnCl 15 Two timesthe amount of NaNO;, and two times the amount of NH NO I 28 Omit Mg++ 80Add Mn++ as MnCl replacing KCl 101 Replace Mg++ with Mn++ 80 Replaceyeast with two times the amount of NaNO 57 Four times the amount ofyeast and two times the amount of NaNO, 89 Four times the amount ofyeast, two times the amount of NaNO and two times the amount of NH NO 92'Replace 'Fe++ with Mn 8O Replace Fe with Mn++ 8-8 EXAMPLE IVFermentations A through C were carried out in the manner described inExample II with variation in the grade of exfoliated vermiculite used.The results of the production of F.E.S. at four weeks is as follows:

Fermentation A 4 wks.(g,/tray) Control, as in Example II 165 Terralite,Sp. Gr. #2, an agricultural size Grade 2, as identified in Table IIdusted to have a good particle size 181 Terralite, gr. #4 as identifiedin Table II 180 Terralite, gr. #3 as identified in Table II .I... 184Bar B base, large size vermiculite, less than number 1 US. sieve sizemade with a low exfoliation temp. Substrate, exfoliated vermiculite witha high bulk density on the order of Bar B base 68 Fermentation BTerralite, gr. #4 see Table II 173 Terralite, gr. #3 see Table II 183Control 161 Verxite, gr. #4 see Table II 136 Terralite, gr. #4 see TableII 214 Fermentation C Control 151 Verxite, gr. #3 see Table II 194Terralite, gr. #3 see Table II 184 Verxite, gr. #4 see Table II 192Terralite, gr. #4 see Table II 178 EXAMPLE V Fermentations D through Iwere performed using the procedure of Example II with variation in thecarbohydrate source and Water.

Fermentation D Control (glucose, 37%) Sucrose, 37% 234 Fermentation EControl,Tray #1 (glucose, 37%) Control, Tray #2 (glucose, 37%) -1. 176Sucrose, 37% 237 Fermentation F Control (glucose, 37%) I- 114 Sucrose,37% 147 Sucrose, 30% 148 Fermentation G Control (glucose, 37%) 134Sucrose, 37% 190 Maltose, 37% '83 A 1:1 mixture of sucrose and maltose,37% 119' Fermentation H Grams/tray for V 4 weeks 5 weeks Sucrose, dist.H20 196 222 Sucrose, dist. H2O 172 228 Sucrose, tap H20 156 Sucrose, tapH2O 142 198 Fermentation I Sucrose, dist. H2O 192 222 Sucrose, dist. H2O213 218 Sucrose, deionized H2O I. 219 251 Sucrose, deionized H2O 208 246EXAMPLE VI The following table illustrates utilization of differentamounts of production medium used in fermentations J and K carried outon trays according to the procedure of Example II.

Fermentation J It is claimed:

1. A synthetic fermentation medium, especially for use in cultivation ofthe microorganism Gibberellal zeae, consisting essentially of a supportof exfoliated vermiculite having a particle size between about sievenumber 1 and sieve number 100, US. Sieve Series, and a bulk density ofbetween about 4 and 12 pounds per cubic foot and an aqueous nutrientmedium containing assimilable carbohydrate source and assimilablenitrogen source, said carbohydrate source being a sugar and being'present in solution in the nutrient medium in an amount sufficient forreduction by the microorganism but below the amount at which the sugarbegins to crystallize out of the medium.

2. The medium of claim 1 wherein the nutrient medium is included in aratio of about 240 milliliters for about 40 to 240 grams of theexfoliated vermiculite.

3. The medium of claim 1 wherein the sugar is selected from the groupconsisting of glucose, sucrose and maltose. v

4. The medium of claim 3 wherein the nutrient medium is included in aratio of about 240 milliliters for about 40 to 240 grams of theexfoliated vermiculite.

5. The medium of claim 4 wherein the nutrient medium contains potassium,phosphorus, sodium and nitrogen ions, each in an amount sufficient toinsure proper growth of the microorganism.

6. The medium of claim 3 wherein thhe vermiculite has a bulk density ofbetween about 5 and pounds per cubic foot.

7. The medium of claim 3 wherein the vermiculite has a bulk density ofbetween about 5 and 7 pounds per cubic foot.

8. The medium of claim 3 wherein the nutrient medium contains potassium,phosphorus, sodium and nitrogen ions, each in an amount suflicient forgrowth of the microorganism.

9. The medium of claim 8 wherein the amounts of ions are between about0.001 and 1% by weight of the nutrient medium.

10. A synthetic fermentation medium, especially for use in cultivationof the microorganism Gibberella zeae, consisting essentially of asupport of exfoliated vermiculite having a particle size between aboutsieve number 1 and sieve number 100, US. Sieve Series, and a bulkdensity in the range of about 5 to 7 pounds per cubic foot and anaqueous nutrient medium containing about 30 to 45 percent of dissolvedsugar selected from the group consisting of glucose, sucrose and maltoseand about 0.001 to 1 percent of assimilable potassium, phosphorus,sodium and nitrogen ions, said percentages being based on the weight ofthe nutrient medium, and said support being present in the fermentationmedium in a ratio of about 40 to 240 grams per each 240 milliliters ofsaid nutrient medium.

11. The medium of claim 10 wherein the support of exfoliated vermiculiteis arranged in a bed having a depth of about 1 to 6 inches.

12. In a process for the production of the compound by cultivation ofthe microorganism Gibberella zeae, the improvement of cultivating themicroorganism in a synthetic fermentation medium consisting essentiallyof a support of exfoliated vermiculite having a particle size betweenabout sieve number 1 and sieve number 100, US. Sieve Series, and a bulkdensity of between about 4 and 12 pounds per cubic foot and an aqueousnutrient medium containing assimilable carbohydrate source andassimilable nitrogen source, said carbohydrate source being a sugar andbeing present in solution in the nutrient medium in an amount snfiicientfor reduction by the microorganism to produce said compound but belowthe amount at which the sugar begins to crystallize out of the medium,said cultivating being for a period of time 10 sufiicient to produce thecompound, and thereafter recovering the compound.

13. The process of claim 12 wherein the sugar is selected from the groupconsisting of glucose, sucrose and maltose.

14. The process of claim 13 wherein the vermiculite has a bulk densityof between about 5 and 10 pounds per cubic foot.

15. The process of claim 14 wherein the nutrient medium is included in aratio of about 240 milliliters for about 40 to 240 grams of theexfoliated vermiculite.

16. The process of claim 15 wherein the cultivation is carried out at atemperature of about 12 to 25 C.

17. The process of claim 16 wherein the temperature is about 15 to 17 C.

18. The process of claim 12 wherein the cultivation is carried out at atemperature of about 14 to 19 C.

19. The process of claim 16 wherein the microorganism is the Gibberellazeae strain 'NKRL 3311.

20. In a process for the production of the compound by cultivation ofstrain NRRL 3311 of the microorganism Gibberella zeae, the improvementof cultivating the microorganism at a temperature of about 15 to 17 C.,in a synthetic fermentation medium consisting essentially of a supportof exfoliated vermiculite having a particle size between about sievenumber 1 and sieve number US. Sieve Series, and a bulk density in therange of about 5 to 7 pounds per cubic foot and an aqueous nutrientmedium containing about 30 to 45 percent of dissolved sugar selectedfrom the group consisting of glucose, sucrose and maltose, yeast andabout 0.001 to 1 percent of assimilable potassium, phosphorus, sodiumand nitrogen ions, said percentages being based on the weight of thenutrient medium, and said support being present in the fermentationmedium in a ratio of about 40 to 240 grams per each 240 milliliters ofsaid nutrient medium, said cultivating being for a period of timesufiicient to produce the compound, and thereafter recovering thecompound.

21. The process of claim 20 wherein the support of exfoliatedvermiculite is arranged in a bed having a depth of about 1 to 6 inches.

References Cited UNITED STATES PATENTS 3,028,312 4/1962 Le Mense 1953,196,019 7/1969 Andrews et al. 992 3,373,025 3/1968 Hodge et al. 99-2LIONEL M. SHAPIRO, Primary Examiner US. Cl. X.R. 195-36; 116

